Simple and Sensitive Method for Determination of Protein Kinase

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A simple and sensitive method for determination of protein kinase activity based on surface charge change of peptide-modified gold nanoparticles as substrates Fang Yi, Xiangyi Huang, and Jicun Ren Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04569 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Analytical Chemistry

A simple and sensitive method for determination of protein kinase activity based on surface charge change of peptidemodified gold nanoparticles as substrates Fang Yi, Xiangyi Huang*, Jicun Ren* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China. ABSTRACT: Protein tyrosine kinases play a pivotal role in intracellular signal transduction pathways and oncogenic transformation. It is necessary to develop a simple, cost-effective and sensitive kinase assay for study of protein kinases and discovery of kinase-target drugs. In this paper, we present a simple and sensitive method for homogeneous detection of protein kinase activity and screening of inhibitor by measuring surface charge change on the peptide-modified gold nanoparticles (GNPs) as kinase substrates. In this assay, Abl (Abelson murine leukemia viral oncogene) kinase was used as a model. In the presence of Abl kinase and ATP, the surface negative charge on GNPs significantly increases due to phosphorylation of the peptide-modified GNPs. The surface charge on the peptide-modified GNPs was measured by zeta potential analyzer. Under the optimum conditions, the zeta potential on the peptide-modified GNPs was linearly dependent on Abl kinase concentration, the linear range was from 1 to 40 nM and the detection limit was 1 nM. This method was used to evaluate the inhibition efficiency of inhibitors, and the obtained IC50 values were well in agreement with the results reported in the references. Furthermore, this method was successfully applied to determine Abl kinase activity in the cell lysates. Compared to current methods, this new method shows simplicity, short analysis time, high sensitivity, and will become a promising platform for kinase-related fundamental research and inhibitor screening.

Protein tyrosine kinases (PTKs) are charged with protein phosphorylation and play a pivotal role in intracellular signal transduction pathways and oncogenic transformation.1 Abnormal expression and aberrant phosphorylation of PTKs are responsible for various diseases, such as cancer, diabetes, Alzheimer’s disease, immune deficiencies, and inflammatory diseases.2-6 For example, Abl (Abelson murine leukemia viral oncogene) is a kind of protein tyrosine kinases. It is associated with the growth and differentiation of hematopoetic cells, and it can promote the occurrence and progression of cancer when Chronic Myeloid Leukemia (CML) fusion protein Bcr-Abl is not regulated.7 Therefore, PTKs are considered as very important molecular targets for the pharmaceutical therapy of several diseases. It is necessary to develop a simple, sensitive and low-cost method for determination of the protein kinase activity and screening of inhibitors. So far, some kinase inhibitors have been used to suppress the activities of related PTKs. Radiometric method represents the conventional assay of kinase activity using γ-phosphate (generally 32P or 33P) as the substrate.8,9 This method is very sensitive, but it needs the complicated multistep processes and use of harmful radioactive materials. In order to avoid radioactive contaminations, certain methods have been developed for kinase assays and screening inhibitors, including mass spectrometry,10-12 electrochemistry,13-16 optical methods,17-19 colorimetric methods,20 and fluorescence methods21-26 based on fluorescence intensity assays, fluorescence polarization assays, fluorescence resonance energy transfer (FRET), and timeresolved fluorescence. Most of these techniques have the advantages of high throughput and high sensitivity.27 But these

methods usually require expensive instruments or complicated surface immobilization and rinsing processes.28 Recently, gold nanoparticles (GNPs) as labeling probes have been widely used for bioassay and bioimaging due to their unique optical properties, good biocompatibility, and ease of chemical modification.29-31 Some strategies for combination of colorimetric18,32-35 and electrochemical techniques36,37 with GNPs as probes, have been developed for determination of protein kinases and screening of inhibitors. Brust et al. developed a simple colorimetric protocol for detection of kinase activity and inhibition.18 In this assay, peptide-capped GNPs were used as kinase substrates and γ-biotin-ATP was used as an ATP mimic. In the presence of kinase, peptide-capped GNPs were phosphorylated and the phosphorylated peptidecapped GNPs with biotin were bound to avidin-modified GNPs, which resulted in a marked color change. Wang et al.37 proposed a highly sensitive electrochemical biosensor for the detection of kinase activity based on the DNA and GNPs induced signal amplification. Although the above methods are effective and highly sensitive, they need tedious modification of GNPs, costly labeled substrates and time-consuming for observing the color changes.35 Additionally, certain factors, such as the surface states of GNPs and uncontrolled aggregations of GNPs, maybe have an impact on the analytical results. Therefore, it is still highly desirable to develop simple, sensitive and low-cost methods for determination of protein kinases and screening of inhibitors. Corresponding Author

* Phone: +86-21-54746001. Fax: +86-21-54741297. Email: [email protected]

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The zeta potential measurement is usually used to characterize the stability and the surface functionalization of nanoparticles.20,38-41 Cho et al.40 synthesized a covalently functionalized fluorescent polymeric nanoparticles (NPs) library and evaluated the role of surface charge on the acute inflammation and the localization in the lung. Their results implied that surface charge (or zeta potential) of functionalized polymeric NPs was a key factor influencing lung inflammation. Furthermore, the zeta potential was also used to monitor the surface functionalization of NPs.20, 39 To the best of our knowledge, so far there are no reports about studying the quantitative relationship between the zeta potentials of NPs and chemical properties of biomolecules and detecting activities of protein kinases. Herein we for the first time report a simple and sensitive method for the homogeneous detection of tyrosine kinase activity and screening of its inhibitors based on the measurement of zeta potential. The principle of this new method is based on the determination of the surface charge change on the peptidemodified GNPs as substrates during phosphorylation. In this assay, Abl kinase was used as an illustrative example. The peptide-modified GNPs as kinase substrates can be phosphorylated in the presence of Abl kinase and ATP. In the phosphorylation, the surface negative charges of GNPs increased, resulting in an increase in the zeta potential of GNPs. We found that the change in the zeta potential of GNPs was associated with Abl kinase concentration at optimal conditions. This method was used to determine the activity of Abl kinase and evaluate the inhibition efficiency of inhibitors. The obtained IC50 values were well in agreement with the results reported in the literature. Furthermore, this method has been successfully applied for determination of kinase activity in the cell lysates. EXPERIMENTAL SECTION Chemicals and Materials. Abl protein tyrosine kinase was supplied by Sino Biological Inc (Shanghai, China). Substrate peptide (sequence, EAIYAAPFAKKK) of Abl was synthesized by GL Biochem (Shanghai, China). HAuCl4·4H2O, sodium citrate, and sodium orthovanadate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). ATP, Bis-(p-sulfonatophenyl) phenylphosphine dehydrate dipotassium (BSPP), 4, 7, 10, 13, 16, 19, 22, 25, 32, 35, 38, 41, 44, 47, 50, 53 - Hexadecaoxa - 28, 29 dithiahexapentacontanedioic acid di-N-succinimidyl ester (NHS-PEG-S-S-PEG-NHS, C46H80N2O24S2, MW: 1109.26) and O - [2-(3-Mercaptopropionylamino) ethyl]- O′ methylpolyethylene glycol (HS-PEG-CH3, CH3O(CH2CH2O)nCH2CH2SH, MW: 5000) were products of Sigma-Aldrich (St. Louis, MO, U.S.A.). The tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib were obtained from Ange Pharmaceutical Co., Ltd (Nanjing, China). Human K562 erythroleukemic cells were from American Type Culture Collection (ATCC, USA). 1 × glo-lysis was supplied from Promega Biotech Co., Ltd (Beijing, China). The improved Bradford protein assay dye reagent kit was from Sangon (Shanghai, China). All other reagents used in this work were of analytical quality. Ultrapure water (18.2 MΩ cm) from the Millipore Simplicity system (Millipore, Bedford, MA) was used throughout the experiments. UV-Vis absorption spectra of GNPs were obtained on a UV-3501 spectrophotometer (Tianjin Gangdong Science & Technology Development Co. Ltd., China). Transmission electron microscopy (TEM) measurements were taken with a JEOL, 2010F, Japan. The conju-

gates of NHS-PEG disulfide and peptides were characterized by Nano-Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometer (NanoLC/Q-TOFMS, Bruker Daltonics Inc, Germany). Resonance light scattering correlation spectroscopy (RLSCS) was used to characterize GNPs and GNPspeptide conjugates. RLSCS measurements were performed on a home-built RLSCS system, and the details of the experimental setup can be found elsewhere.42 Zeta potential measurement was recorded with a Nano Zetasizer ZS90 (Malvern, U.K.). Synthesis of GNPs. About 17 nm GNPs used in this work were synthesized based on a reported method.43 Briefly, all glass used in the following procedure was cleaned in aqua regia (3:1 HCl-HNO3), rinsed with water thoroughly. 95 mL water contained with 1mL of HAuCl4 (1%, w/w) was heated to reflux under stirring, and then 5 mL of sodium citrate solution (1%, w/w) was added to the boiling HAuCl4 solution rapidly, which resulted in a color change from pale yellow to wine red. After the color changed, the colloidal solution was kept stirring for 15 min at room temperature and finally stored at 4 °C for further use. Preparation of functionalized GNPs. The conjugation approach is similar to the reported procedure. 44 Briefly, 2 mL of GNPs was centrifuged at 6500 rpm for 25 min and resuspended in 150 µL of 1 mg mL-1 BSPP solution. 12 µL NHS-PEG-S-S-PEG-NHS (2.5 mM) and 15 µL substrate peptide (0.4 mM) was firstly mixed with 40 µL of borate buffer (50 mM, pH 8.2, 25 °C) with gently stirred for 3 h. Then the resulting solution was introduced to the GNP dispersions and reacted for another 3 h. Finally, 10 µL HS-PEG-CH3 (4 mM) was added to the mixture in order to enhance the stability of GNPs and kept stirred for additional 5-8 h. The mixture was centrifuged at 6500 rpm for 30 min to remove unreacted chemicals and re-suspended in 15 mM Tris-HCl buffer (pH 7.5, 25 °C). Standard procedures for detection of Abl activity and inhibition study. Typically, 200 µL of the Abl reaction solution composed of about 10 nM peptide-conjugated GNPs, the varied concentrations of Abl (1-100 nM), 5 mM MgCl2, 0.2 mM ATP, and 15 mM Tris-HCl buffer were incubated for 60 min at 30 °C. For Abl inhibition assay, inhibitors dissolved in DMSO were diluted to different concentrations in ultrapure water, mixed with 40 nM Abl, and then were added into the reaction solution. The following experiment procedures were carried out under the above-mentioned conditions. Zeta potentials of these solutions were measured in 15 mM Tris-HCl buffer (pH 7.5, 25 °C) and recorded with a Nano Zetasizer ZS90. The final GNPs concentration was about 3 nM. Determination of Abl activity in K562 cell lysate. Human K562 cells cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Corning), 100 mg mL-1 of streptomycin, and 100 U mL-1 of penicillin in a humidified 5% CO2 atmosphere at 37 °C. K562 cells were pretreated with sodium orthovanadate to inhibit phosphatases.45 After 30 min of stimulation, the cultured cells were centrifuged (1000 rpm, 4 min) to remove the medium. Then the cells were washed with phosphate buffer, and re-suspended in cell lysis buffer (1 × glo-lysis buffer, pH 7.5). The cell lysate was incubated on ice for 15 min and centrifuged at 12,000 rpm for 5 min at 4 °C. The extracted supernatant was diluted to an appropriate concentration in the following assay. The total protein concentration of cell lysate was determined by the Bradford method using bovine serum albumin (BSA) as the standard. Briefly,


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Analytical Chemistry different concentrations of BSA (5-30 µg mL-1) were mixed with the Bradford reagent for 10 min. The absorbance of each standard was measured at 595 nm using a UV-Vis spectrophotometer, and the standard curve was obtained by linear regression. Finally, the cell samples were mixed with Bradford reagent, measured by the above mentioned. The cell lysate was diluted to 20 µg mL-1 in the following experiment. For phosphorylation reactions with cell lysates, the same procedures were performed except the cell lysate (20 µg mL-1, with different degrees of simulation) above prepared instead of purified Abl kinase. Negative control samples were prepared with unstimulated cell lysates or stimulated cell lysates (treated by sodium orthovanadate) in the absence of ATP or in the presence of kinase inhibitors. The zeta potentials of these solutions were recorded with a Nano Zetasizer ZS90. RESULTS AND DISCUSSIONS Principle of protein kinase assay. The principle of the protein kinase assay is based on the determination of the surface charge change on the peptide-modified GNPs as kinase substrates before and after phosphorylation. The assay procedure is illustrated in Scheme 1. In this study, Abl kinase is used as a model and the peptide-modified GNPs are used as Abl kinase substrates containing the phosphorylation site Tyr. In the presence of kinase and ATP, peptide-modified GNPs are phosphorylated, which results in an increase in their surface negative charge. The zeta potential analyzer can be used to conveniently measure the surface charge of GNPs. During the phosphorylation of substrates, the zeta potential of GNPs increases with an increase in the Abl kinase concentration. Therefore, we can quantify the Abl kinase activity and screen its inhibitors according to the relationship between the zeta potential of GNPs and the concentration of Abl kinase.

Scheme 1. The principle of the kinase activity assay using peptide-modified GNPs as substrates.

Preparation of peptide-modified GNPs. Because the substrate peptides used in this study are positive charge, they are easy to aggregate with negatively charged GNPs through electrostatic attraction when only the substrate peptides are used for the modification of GNPs. The important consideration is to increase the stability of the peptides functionalized GNPs. In our former study,39 we found that PEG-SH as ligands sig-

nificantly improved the stability of GNPs. Hence we tried to use PEG-SH as ligands to enhance the stability of GNPs in this study. The substrate peptides were firstly conjugated with NHS-PEG disulfide via the reaction between succinimidyl ester and amino. The conjugates of PEG disulfide and peptides have been characterized by LC-MS and the results showed that the substrate peptides were conjugated with NHS-PEG disulfide successfully (shown in Figure S1). Then, GNPs were modified with substrate peptides via Au-S bond due to high affinity of thiol compounds for the GNPs. Furthermore, HSPEG-CH3 was connected to the GNPs surface via Au-S bond, which increased the stability of GNPs as well as suppressed the nonspecific binding of the GNPs surface.39 The average amount of peptide modified on GNPs was quantified by the fluorescence-based assay.44 In this assay, GNPs were modified by FITC conjugated substrate peptides (sequence, EAIYAAPFAKKK - FITC). We found that the peptide to GNPs ratio is estimated to be about 200:1, which is similar to that of the report.44 The prepared GNPs were characterized by TEM, Zeta analyzer, and UV-Vis spectrophotometer. Figure 1A shows the TEM image of 17.0 ± 4.3 nm GNPs. As shown in Figure 1B, the zeta potential of unmodified GNPs is -29.0 ± 0.7 mV, while the zeta potential of substrate peptide-conjugated GNPs is -6.0 ± 0.4 mV. The large change in zeta potential is mainly attributed to the fact that the substrate peptide is positively charged and PEG-SH is a neutral polymer in aqueous solution. The result illustrated that the GNPs were successfully modified with substrate peptide and HS-PEG-CH3. Figure 1C displays the UV-Vis absorption spectra of GNPs before and after the substrate peptide conjugation, and about 5 nm red-shift in the plasmon resonant absorption peak produced after the modification process.

Figure 1. (A) The TEM image of 17 nm GNPs, the scale bar is 50 nm. (B) Zeta potentials of GNPs before and after the substrate peptide conjugation. (C) UV-Vis absorption spectra of GNPs before (blue) and after (red) the substrate peptide conjugation. (D) The RLSCS auto-correlation normalized curves of GNPs (blue) and peptide-conjugated GNPs (red).

Furthermore, resonance light scattering correlation spectroscopy (RLSCS) is further applied to characterize the conjugation of GNPs with peptides. The principle of RLSCS is similar to fluorescence correlation spectroscopy (FCS) and it is based on the fluctuation of the resonance light scattering of



metal nanoparticles such as GNPs in small detection volume due to the Brownian motion of single nanoparticles.42 The auto-correlation normalized curves of GNPs before and after the peptide conjugation are shown in Figure 1D. The characteristic diffusion time of GNPs and peptide-conjugated GNPs were determined as 0.63 ms and 0.92 ms, respectively. The data reflected that the hydrodynamic radius of peptideconjugated GNPs was bigger than that of GNPs. These results above further illustrated that the GNPs were successfully conjugated with substrate peptides. Then the stability of modified GNPs was investigated in NaCl solutions with different concentrations (0, 5, 10, 20, 40, and 80 mM). The UV-Vis spectra of modified GNPs are shown in Figure S2, suggesting that peptide-conjugated GNPs are stable enough in some certain environments.

Figure 2. (A) Effects of the ratio of substrate peptide to GNPs on the zeta potential. (B) Effects of the ratio of HS-PEG-CH3 to GNPs on the zeta potential. S-peptide: substrate peptide, Ppeptide: phosphorylated peptide. The zeta potentials were the average of three replicates for each data point, and the error bars were obtained from the standard error of means.

Optimization of experimental conditions. The mole ratios of substrate peptides and HS-PEG-CH3 to GNPs were systematically optimized in order to enhance the zeta potential change of GNPs before and after the phosphorylation. Firstly, the peptide-conjugated GNPs are prepared by mixing GNPs and peptides in different mole ratios (500:1, 1000:1, 1500:1, 2000:1, and 2500:1) of peptides to GNPs, and the concentration of GNPs is about 2.0 nM. The change of zeta potential of the peptide-conjugated GNPs is tested in phosphorylation, and the results are shown in Figure 2A. The optimized mole ratio of peptide to GNPs is found to be 1500:1 since the zeta potential change of GNPs was the biggest before and after the phosphorylation. Furthermore, the mole ratio of HS-PEG-CH3 to GNPs is optimized, and the best ratio of HS-PEG-CH3 to GNPs is 10000:1 (as shown in Figure 2B). Therefore, these ratios of substrate peptide and HS-PEG-CH3 to the GNPs were

used in the subsequent experiments. Finally we optimized the concentration of GNPs and found that the zeta potential change of GNPs was bigger before and after the phosphorylation when the concentration of GNPs was increased. However, as shown in Figure S3, the system is unstable and the error bar is bigger when 6 nM GNPs were used. So the concentration of 3 nM GNPs was chose for further experiments because of bigger zeta potential change and better stability of the system. Determining Abl activity and evaluating inhibition efficiency of drugs. The determination of Kinase Ab1 activity was carried out under the above optimized conditions with different concentrations of Abl (1-100 nM). Figure 3 shows that the zeta potentials of peptide-conjugated GNPs increase significantly with the increase of Abl concentrations, and then reached a platform when Abl concentration was 40 nM. A linear relationship between the zeta potentials (Y) and the concentrations of Abl (CAbl (nM)) was obtained from 1 to 40 nM and can be expressed as Y = -5.37 - 0.0996 CAbl (the correlation coefficients R2 = 0.993). The detection limit of Abl is 1 nM, which is comparable or more sensitive than those of PTK assays based on polyion complex nanoparticles amplification, quantum dots amplification and electrochemical measurements.22,23,36

Figure 3. The relationship between zeta potentials and Abl concentrations. The inset is the linear relationship curve between zeta potentials and Abl concentrations. The zeta potentials were the average of three replicates for each data point, and the error bars were obtained from the standard deviation of three replicates. Experiment conditions are the same as above mentioned in the Experimental Section.

To demonstrate whether this method can be suitable for evaluating the inhibition efficiency of Abl inhibitors, the assay procedure above was used in the presence of the Abl inhibitors with different concentrations. As shown in Figure 4A, the zeta potential of GNPs decreases with the increase in the imatinib concentration. The corresponding zeta potential of GNPs reaches a platform while imatinib concentration is up to 20 µM. The IC50 value of imatinib is 6.34 µM, which is in accordance with that reported in the literature.46 Imatinib is the first generation of tyrosine kinase inhibitors, and it can inhibit Abl activity by targeting the ATP binding site in the catalyst domain of Abl.47 Although imatinib was an important drug for


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Analytical Chemistry the targeted therapy of CML, patients show imatinib resistance eventually during a significant fraction of disease progression.48 In order to overcome imatinib resistance, two kinds of drugs have been approved for the therapy of CML, nilotinib (the 2-phenylaminopyrimidine AMN107)49 and dasatinib (the 2-aminothiazole BMS-354825).50 Furthermore, we investigated the inhibition of nilotinib and dasatinib to Abl activity, and the results are shown in Figure 4B and Figure 4C. The IC50 values of nilotinib and dasatinib were 7.87 nM and 0.34 nM, respectively. Figure 4D shows the inhibition curves of Abl activity by imatinib, nilotinib and dasatinib using SPSS (Statistical Product and Service Solutions) fitting. Table S1 summarized the IC50 values of three inhibitors, and the obtained results are basically in same tendency to IC50 values measured by other methods.48-51 These results demonstrate that this method is suitable for screening of protein kinase inhibitors.

Figure 4. The relationship between the zeta potential and the concentration of Abl inhibitor (A) imatinib, (B) nilotinib, (C) dasatinib, (D) Inhibition of Abl activity by dasatinib (red), nilotinib (green), imatinib (blue), respectively. Inhibition = △Zeta / △Zeta max, △Zeta = Zeta - Zeta0, △Zeta max = Zeta max - Zeta0. The error bars present the standard error of means.

Detection of Abl activity in cell lysate. The Abl kinase has been involved in tumorigenesis and in many important cellular processes, and is considered as an important biomarker for the diagnostics and prognostics of cancers.7 In this study, the drug-induced activities of Abl kinase in human K562 erythroleukemic cell lysates are measured and the results are shown in Figure 5. It is reported that Abl in human erythroleukemic cells is activated by stimulation with a tyrosine phosphatase inhibitor.41 In this case, human K562 erythroleukemic cells were stimulated with various concentrations of sodium orthovanadate. Briefly, the stimulated cell lysates (the total protein concentration was estimated to 20 µg mL-1) were added to GNPs solution in the presence of substrate peptide, ATP and Abl inhibitor dasatinib. Figure 5A shows the corresponding zeta potentials of GNPs with various concentrations of sodium orthovanadate. The change of stimulant concentrationdependent zeta potential indicates that Abl activity in cell lysates gradually increased with increasing sodium orthovanadate concentration, and then slightly decreased after reaching the maximum (0.2 mM). As shown in Figure 5B, the zeta potentials of GNPs are -14.9 mV, -14.5 mV, -13.6 mV and -13.0

mV for lysates of cells treated with 0.5, 1.0, 1.5, and 2.0 nM dasatinib, respectively. Even in the presence of 0.5 nM dasatinib, the zeta potential for lysate from cells treated with the drug was clearly different from that for control cells. Although cell lysate is very complicated, this method was successfully used for detection of the activity of Abl in its inhibitor dasatinib-treated cell lysate. To further verify the reproducibility of this method, we repeated the experiments by conducting several independent assays on different days; the RSDs of zeta potentials are approximately 3.8 % to 9.8 %. These results indicate our method enables reliable Abl activity assay of drug-induced inhibition in cell lysates.

Figure 5. (A) The relationship between zeta potential of peptideconjugated GNPs and the concentration of sodium orthovanadate in K562 cell lysates. (B) The relationship between zeta potential of peptide-conjugated GNPs and the concentration of dasatinib in K562 cell lysates. The concentration of sodium orthovanadate is 0.2 mM. The total protein concentration is estimated to 20 µg mL1 and other conditions are the same as above mentioned in the Experimental Section.

CONCLUSIONS In this paper, we proposed a new method for detecting protein kinase activity and screening kinase inhibitors based on the zeta potential change on peptide-modified GNPs as substrates. To the best of our knowledge, the zeta potential measurement is the first time applied to detect protein kinase activity and screen kinase inhibitors. In this assay, Ab1 kinase is used a model and the peptide-modified GNPs are prepared as phosphorylation substrates. We found that the change in the zeta potential of GNPs is linearly proportional to Ab1 kinase concentration at optimal conditions. The linear range of this method is from 1 to 40 nM and the detection limit is 1 nM. This method was successfully used to determine kinase activi-



ty in the cell lysates and screen kinase inhibitors. Some wellknown methods such as combination of colorimetric, fluorescence and electrochemical techniques with GNPs as the probes, have been developed for determination of protein kinases and screening of inhibitors. The above methods are effective and highly sensitive, however, they need tedious modification of GNPs, costly labeled substrates and timeconsuming for observing the color changes. Additionally, certain factors, such as the surface states of GNPs and uncontrolled aggregations of GNPs, maybe have an impact on the analytical results. Compared with these methods, this new method possesses certain advantages, such as simple, rapid, low-cost, and homogenous without elaborate washing steps and complicated peptide labeling. More importantly, the zeta potential changes on peptide-modified GNPs can be easily measured without expensive instruments. Besides widely used optical and colorimetric sensors, this study provides another kind of biosensors for protein kinases. We believe that this method may become a promising platform for kinase assay in realistically complex systems and rapidly screening kinaserelated drugs. Our method for combination of zeta potential change with PNA-modified GNPs as probes, may also be developed for the determination of nucleic acids since the negative charges on the surface of GNPs increased significantly after the hybridization.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details for the characterizations of the substrate peptides conjugated with NHS-PEG disulfide and the average amount of peptides modified on GNPs, as well as Supplementary figures, tables, and references (PDF).

AUTHOR INFORMATION Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Science Foundation of China (21675111, 21327004), and the Shanghai Natural Science Foundation (14ZR1423400).

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